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Endocrinology, doi:10.1210/en.2004-1145
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Endocrinology Vol. 146, No. 2 532-543
Copyright © 2005 by The Endocrine Society

A Fully Functional Proopiomelanocortin/Melanocortin-1 Receptor System Regulates the Differentiation of Human Scalp Hair Follicle Melanocytes

Söbia Kauser, Anthony J. Thody, Karin U. Schallreuter, Christopher L. Gummer and Desmond J. Tobin

Department of Biomedical Sciences, University of Bradford (S.K., A.J.T., K.U.S., D.J.T.), Bradford BD7 1DP, United Kingdom; and Procter and Gamble Ltd. (C.L.G.), Surrey TW20 9NW, United Kingdom

Address all correspondence and requests for reprints to: Prof. Desmond J. Tobin, Department of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire BD7 1DP, United Kingdom. E-mail: d.tobin{at}bradford.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The proopiomelanocortin (POMC)-derived peptides, ACTH and {alpha}-MSH, are the principal mediators of human skin pigmentation via their action at the melanocortin-1 receptor (MC-1R). Recent data have demonstrated the existence of a functionally active ß-endorphin/µ-opiate receptor system in both epidermal and hair follicle melanocytes, whereby ß-endorphin can regulate melanogenesis, dendricity, and proliferation in these cells. However, a role for ACTH and {alpha}-MSH in the regulation of the human follicular pigmentary unit has not been determined. This study was designed to examine the involvement of ACTH and the {alpha}-MSH/MC-1R system in human follicular melanocyte biology. To address this question we employed RT-PCR and immunohisto/cytochemistry, and a functional role for these POMC peptides was assessed in follicular melanocyte cultures. Human scalp hair follicle melanocytes synthesized and processed POMC. ACTH and {alpha}-MSH in association with their processing enzymes and MC-1R are expressed in human follicular melanocytes at the message level in vitro and at the protein level both in situ and in vitro. The expression of the POMC/MC-1R receptor system was confined only to subpopulations of poorly and moderately differentiated melanocytes. In addition, functional studies revealed that ACTH and {alpha}-MSH are able to promote follicular melanocyte differentiation by up-regulating melanogenesis, dendricity, and proliferation in less differentiated melanocyte subpopulations. Thus, these findings suggest a role for these POMC peptides in regulating human hair follicle melanocyte differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACTH AND {alpha}-MSH are cleaved from the precursor prohormone proopiomelanocortin (POMC) by the action of prohormone convertase 1 (PC1) and PC2, respectively. Other peptides, including ß-lipotropic hormone and ß-endorphin (ß-END), are also produced from POMC (1, 2).

The involvement of ACTH and {alpha}-MSH in human skin pigmentation was first recognized by the stimulation of melanogenesis upon systemic administration of ACTH, {alpha}-MSH, and ß-MSH, especially in sun-exposed regions of the body (3, 4). Additional evidence suggesting their involvement in cutaneous pigmentation comes from several clinical observations; for example, elevated circulating levels of ACTH and {alpha}-MSH or prolonged therapeutic administration of ACTH induce hyperpigmentation in humans (5, 6).

It is now well established that the skin is a local source and target for POMC-derived peptides (5, 7, 8, 9). ACTH, {alpha}-MSH (10), and ß-END (11) are secreted by epidermal keratinocytes in vitro, and all three of the above POMC peptides are also produced by normal human epidermal melanocytes (EM) in vitro (12). The production of ACTH and {alpha}-MSH has also been reported to be up-regulated in response to ultraviolet B radiation in normal human EM and epidermal keratinocyte (13).

{alpha}-MSH and ACTH peptides are proposed to be the principal mediators of human skin pigmentation. They are involved in the regulation of human epidermal melanogenesis, dendricity, and proliferation via action at the melanocortin-1 receptor (MC-1R) (14, 15, 16, 17, 18, 19, 20). Melanocortin signaling via MC-1R is mediated predominately via the cAMP second messenger system (21, 22). However, accumulating evidence suggests that protein kinase C-dependent pathways, via activation of diacylglycerol, are also involved in the regulation of {alpha}-MSH-induced melanogenesis (23, 24, 25). Recently, protein kinase Cß has been shown to regulate melanogenesis via the direct activation of tyrosinase (26). MC-1R signal transduction may also be coupled to phospholipase C-activated production of inositol triphosphate and diacylglycerol, with subsequent mobilization of intracellular calcium (22).

Compelling evidence suggests that POMC peptides are implicated in the regulation of coat color in many mammalian systems. In rodents and several strains of mice as well as in hamsters, {alpha}-MSH stimulates follicular melanogenesis by preferentially increasing the synthesis of eumelanin over pheomelanin (27, 28, 29, 30, 31). In this respect, administration of {alpha}-MSH to mice stimulates tyrosinase activity at the transcriptional, translational, or posttranslational level depending on the phase of the hair cycle (28, 29, 32, 33).

Furthermore, the expression of POMC and POMC peptides ({alpha}-MSH, ACTH, and ß-END), the processing machinery for POMC and MC-1R in normal murine skin, is confined to specific compartments of the hair follicle and is regulated in a hair cycle-dependent manner (34, 35, 36, 37, 38). Their expression is coupled to early anagen, which coincides with the onset of follicular melanogenesis. These observations suggest that POMC products are the main regulators of follicular pigmentation/hair cycling in the murine system. However, fundamental differences exist in hair cycling and pigmentation between mice and humans; in mice, hair growth is synchronized and proceeds in waves over the body, whereas in humans, a mosaic pattern of asynchronous follicle cycling occurs (39). The length of anagen is also markedly different; it is 8–9 d in mice (40) and lasts up to 10 yr in humans (41). Therefore, what may be important control points in the regulation of pigmentation/cycling in murine models may not necessarily be the case in the human system.

Evidence from humans with inactivating mutations in the POMC gene suggests that POMC peptides such as {alpha}-MSH, ACTH, and ß-END may also play an important role in the regulation of human hair pigmentation. POMC-null mutations in humans result in pale skin and orange/red hair phenotype; this is thought to reflect a lack of ligands for MC-1R (42, 43). Likewise, MC-1R polymorphisms that reduce MC-1R activity have been associated with fair skin and red hair (44, 45).

We have recently shown the existence of a functionally active ß-END/µ-opiate receptor (µ-opiate R) system in human hair follicle melanocytes (HFM), suggesting a potential role for POMC peptides in the regulation of human hair pigmentation. ß-END was shown to be a stimulator of follicular melanocyte melanogenesis, dendricity, and proliferation. Moreover, this system is expressed in follicular melanocytes as a function of their anatomical location and differentiation status during the hair growth cycle (46). However, the role of {alpha}-MSH and ACTH peptides in the regulation of human HFM biology has received little attention to date.

The present study examines the involvement of the {alpha}-MSH-ACTH/MC-1R system in human follicular melanocytes in vitro and in situ. We present evidence that these POMC peptides, in association with their processing enzymes and MC-1R, are differentially expressed in HFM as a function of their anatomical location and activity within the hair follicle, whereby their expression is confined to a subpopulation of melanocytes. In addition, both {alpha}-MSH and ACTH peptides are modulators of follicular melanocyte melanogenesis, dendricity, and proliferation. Thus, {alpha}-MSH and ACTH may have a regulatory role in follicular melanocyte biology.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and culture of hair follicle cell subpopulations of melanocytes
Human haired scalp tissue was obtained with informed consent and local ethics committee approval from seven females (age range, 43–62 yr; mean age, 52 yr; hair color, light brown to black), after elective plastic surgery. All cell culture reagents were obtained from Invitrogen Life Technologies, Inc. (Paisley, Scotland, UK) unless otherwise stated. Skin samples were collected in RPMI 1640 medium and were processed within 5 h of surgery.

HFM cultures were established as described previously (47). Briefly, the epidermis and upper 1 mm of dermis were removed from the scalp specimens. The remaining tissue was incubated in Eagle’s MEM (EMEM) with Earle’s salts and Glutamax-L supplemented with 10% fetal calf serum (FCS), 200 U/ml penicillin, 200 µg/ml streptomycin, 5 µg/ml fungizone, and 0.5% collagenase type V (Sigma-Aldrich Corp., Poole, UK). Hair follicles released by enzymatic digestion were washed repeatedly with PBS until they appeared pure by microscopic examination. Single-cell suspensions were obtained by treatment with 0.05% trypsin and 0.53 mM EDTA for 5–10 min at 37 C.

HFM cultures were established using EMEM supplemented with 4% FCS, 1x concentrated nonessential amino acid mixture, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 5 ng/ml endothelin-1, and 5 ng/ml basic fibroblast growth factor. EMEM was used in combination with keratinocyte serum-free medium supplemented with 25 µg/ml bovine pituitary extract (BPE), 0.2 ng/ml recombinant epidermal growth factor, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Cells were incubated at 37 C in a 5% CO2 atmosphere and were fed every third day. When present in the culture, contaminating fibroblasts were removed by treatment with 150 µg/ml geneticin sulfate (G418) for cycles of 48 h (48). Hair follicle keratinocytes (HFK) were separated from the follicular melanocyte cultures by differential trypsinization. The identity of isolated cells was confirmed by immunophenotyping at passage 1 with the melanocyte lineage-specific marker NKI/beteb against glycoprotein100 (gp100) (49).

HFK
HFK cultures were established by preparing single-cell suspensions from isolated hair follicles as described above. Selective trypsinization of HFM at the primary culture stage enabled successful separation of both follicular melanocytes and keratinocytes. The detached HFM were placed into separate cell culture dishes, leaving behind an essentially pure population of HFK; these were subsequently transferred to keratinocyte serum-free medium alone, which does not support melanocyte growth.

Follicular papilla (FP) fibroblasts (FPF)
FPF were isolated using a simplified recently described method (50). Briefly, anagen hair follicles were isolated by microdissection from normal human haired scalp tissue from five females after elective plastic surgery (age range, 43–62 yr; mean age, 50 yr). Using fine forceps, the hair follicles were gripped gently at the suprabulbar region, and the connective tissue sheath was cut at the level of the stalk. This procedure resulted in externalization of the intact FP from the bulb into the dish. The cells were incubated with RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine at 37 C in a 5% CO2 atmosphere. Culture medium was replenished every third day once cell migration was evident.

Forty-eight hours before all experiments, FCS and/or BPE were omitted form the culture medium for all cell types (follicular melanocytes, keratinocytes, and FPF). This treatment was necessary to ensure the removal of all exogenous sources of POMC peptides, because the half-lives of {alpha}-MSH and ACTH are less than 60 min (28).

Isolation of RNA
Total RNA was isolated from follicular melanocytes, keratinocytes, and FPF by the guanidinium thiocyanate-phenol-chloroform based method, using Tri-Reagent (Sigma-Aldrich Corp.) according to the manufacturer’s instructions. To purify total RNA isolated from HFM, the Dynabeads mRNA direct kit (Dynal Biotech AS, Oslo, Norway) was subsequently used, according to the manufacturer’s instructions. This additional purification step was essential to remove traces of melanin, which can be inhibitory to the PCR (51). Extracted total RNA samples were additionally treated with deoxyribonuclease I (amplification grade; Invitrogen Life Technologies, Inc.) according to the manufacturer’s instructions; this avoided possible contamination of genomic DNA.

RT-PCR
The synthesis of cDNA was performed using RevertAid Moloney murine leukemia virus reverse transcriptase (MBI Fermentas, Vilnius, Lithuania) according to the manufacturer’s instructions using 2 µg total RNA or 0.2 µg mRNA, oligo(deoxythymidine)18, and random hexamer primers (Sigma-Genosys Corp., Pampisford, UK). All PCR reagents were obtained from MBI Fermentas unless otherwise stated. The PCR parameters used for the primers were as follows.

POMC.
POMC was amplified with primer sets, as described previously (52), using a modified protocol: one cycle at 94 C for 5 min, 35 cycles at 94 C for 1 min, 67 C for 1 min, 72 C for 1 min, and a final cycle at 94 C for 1 min, 67 C for 1 min, and 72 C for 10 min.

MC-1R.
MC-1R was amplified with primer sets, as described previously (53), using a modified protocol of touchdown PCR with one cycle at 94 C for 5 min, 10 cycles at 94 C for 1 min 63–58 C (with a decrease of 0.5 C/cycle) and 72 C for 1 min, and 30 cycles at 94 C for 1 min, 58 C for 1 min, and 72 C for 1 min.

PC1.
PC1 was amplified using primer sets and conditions described previously (54): one cycle at 94 C for 10 min, 53 C for 45 sec, and 72 C for 1 min, followed by 33 cycles at 94 C for 45 sec, 53 C for 45 sec, and 72 C for 1 min, and a final cycle at 94 C for 45 sec, 53 C for 45 sec, and 72 C for 10 min.

PC2.
PC2 was amplified using primer sets and conditions described previously (54): one cycle at 94 C for 5 min, followed by 38 cycles at 94 C for 30 sec, 68 C for 45 sec, and 72 C for 45 sec, and a final cycle at 94 C for 30 sec, 68 C for 45 sec, and 72 C for 10 min.

7B2.
7B2 was amplified with primer set, as described previously (54), using a modified protocol of touchdown PCR with one cycle at 94 C for 5 min; 10 cycles at 94 C for 1 min, 64–53 C (with a decrease of 0.5 C/cycle), and 72 C for 1 min; and 30 cycles at 94 C for 1 min, 58 C for 1 min, and 72 C for 1 min.

EM or plasmids containing POMC and MC-1R genes (gifts from Dr. J. Ancans, University of Riga, Riga, Latvia) were used as a positive control for POMC, MC-1R, PC1, PC2, and 7B2 mRNA expression. RNA samples that were not reverse transcribed and omission of cDNA from the reaction mixture served as negative controls.

Amplifications were performed using the Hybaid PCR sprint temperature cycling system (Hybaid, Ashford, UK). Ten microliters of the reaction mixture were mixed with 4 µl gel loading solution (MBI Fermentas) and loaded onto a 1.5% agarose gel (Sigma-Aldrich Corp.) containing 1 µg/ml ethidium bromide (Sigma-Aldrich Corp.); a 100-bp DNA ladder (New England Biolabs, Hitchin, UK) was also loaded. This was followed by electrophoresis at a constant voltage of 100 V using 0.5x Tris-borate buffer. Gels were photodocumented using the UVitec gel documentation system (UVitec Ltd., Cambridge, UK).

Immunohistochemistry
Normal human haired scalp tissue obtained after elective plastic surgery (from five females; age range, 43–60 yr; mean age, 50 yr) or occipital scalp tissue from two normal healthy males (age, 23 and 36 yr) were cryosectioned (7 µm) and processed for double immunolabeling as previously described (55). Briefly, sections were air-dried, fixed in ice-cold acetone, and blocked in 10% normal donkey serum in PBS for 90 min at room temperature. For the detection of MC-1R, sections were fixed in 5% buffered paraformaldehyde for 30 min at room temperature, followed by rinsing with PBS. Sections were incubated with primary antibodies against PC1 (1:200), PC2 (1:200), 7B2 (1:200; PC and 7B2 antibodies were a gift from Prof. N. G. Seidah, Clinical Research Institute of Montréal, Montréal, Canada), {alpha}-MSH (1:50; ICN Biomedicals, Inc., Aurora, CA), ACTH11–39 (1:200; gift from Dr. Y. Peng Loh, National Institutes of Health, Bethesda, MD), and MC-1R (1:50; gift from Dr. M. Böhm, University of Munster, Munster, Germany).

Secondary antibody incubations were performed with a rhodamine-conjugated donkey antirabbit secondary antibody (1:50; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 60 min at room temperature. For the detection of the melanocyte lineage-specific marker gp100, sections were additionally blocked with 10% normal donkey serum and incubated with the second primary antibody, NKI/beteb (1:30; Monosan, Uden, The Netherlands), for 2 h at room temperature and subsequently incubated with a fluorescein-conjugated donkey antimouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) for 60 min at room temperature. Sections were washed in PBS and mounted in Vectashield mounting medium with 4', 6-diamidino-2-phenylindole (Vector Laboratories Ltd., Peterborough, UK). The omission of both primary antibodies, replacement with preimmune serum from secondary antibody host, and inclusion of secondary antibodies served as negative controls. Staining was visualized with a DMIRB/E fluorescence microscope (Leica, Wetzlar, Germany) and photodocumented with the aid of a computer-assisted 3-CCD color video camera (Optivision, Ossett, UK) and the Image Grabber PCI graphics program (Neotech Ltd., Eastleigh, UK). The images produced with the two different fluorochromes, rhodamine (red) and fluorescein (green), were merged using the Paint Shop Pro 7 graphics program (JascSoftware, Oxon, UK). Colocalization of {alpha}-MSH, ACTH, PC1, PC2, and 7B2 with gp100-positive HFM was indicated by the production of a yellow color.

Immunocytochemistry
For staining of cultured follicular melanocytes, keratinocytes, and FPF (passages 2–5), cells were seeded into eight-well Lab-Tek chamber slides (ICN Biomedicals, Inc.) at a density of 5000 cells/well and cultured for 2–3 d . HFM primary cultures were also used, because these cultures contain distinct melanocyte subpopulations, including highly proliferative amelanotic melanocytes that coexist with nonproliferative, intensely pigmented bulbar melanocytes (46, 56).

FCS and BPE were omitted from the culture medium 48 h before immunostaining. Cells were rinsed briefly in PBS for 5 min, then fixed in ice-cold methanol for 10 min at –20 C. For the detection of MC-1R, cells were fixed in 5% buffered paraformaldehyde for 30 min at room temperature, followed by rinsing with PBS. Cells were blocked in 10% normal goat serum rinsed briefly in PBS, and incubated with {alpha}-MSH-, ACTH11–39-, PC1-, PC2-, 7B2-, and MC-1R-specific antibodies at the dilutions described above at 4 C for 18 h. Subsequent steps in immunostaining were performed using the LSAB2 HRP kit and 3-amino-9-ethyl-carbazole substrate system (DakoCytomation, Carpinteria, CA) according to the manufacturer’s instructions or by indirect immunofluorescence, as described above.

Assessment of melanin content, dendricity, and cell proliferation
Follicular melanocyte cultures (n = 7) were grown without FCS and BPE for 48 h before stimulation with acetylated {alpha}-MSH (Ac-{alpha}-MSH; 10–8 M; Bachem, St. Helens, UK) or ACTH 1–17 (10–8 M; Sigma-Aldrich Corp.) for 72 h. A concentration of 10–8 M was chosen for stimulation, because the greatest effects on melanogenesis, dendricity, and proliferation were seen at this concentration with both Ac-{alpha}-MSH and ACTH1–17 during preliminary experiments compared with 10–6 and 10–10 M. Melanocytes from the same donor and at the same passage were maintained in parallel in the absence of the peptide and served as a negative control.

For the assessment of melanocyte dendricity, cells were photographed 72 h after stimulation with Ac-{alpha}-MSH and ACTH1–17. Representative photographs were taken from up to eight random and different fields (of 10 cells for each cell line), and the number of cells with more than three dendrites was counted and compared with controls. After trypsinization, cells were counted using a Neubauer counting chamber. The cells were pelleted by centrifugation, solubilized in 1 M sodium hydroxide, and boiled, and the melanin content was measured spectrophotometrically at 475 nm. A standard curve of synthetic melanin (Sigma-Aldrich Corp.) was the basis for melanin content determination. For each cell line examined, melanin content and cell counts were determined at least three times, and average values were taken. Melanin content was determined as picograms of melanin per cell and expressed as the percent increase in melanin content above that in control unstimulated cells. The increase in cell number was expressed as the percent increase in cell number above control unstimulated levels.

Statistical analysis
Statistical significance was assessed using one-way ANOVA and Dunnett’s posttest.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HFM express MC-1R, POMC, PC1, PC2, and 7B2 mRNA in vitro
RT-PCR with primers specific for MC-1R, POMC, PC1, PC2, and 7B2 produced amplification products of the expected size of 416 bp (MC-1R) and 260 bp (POMC) in normal human follicular melanocytes, keratinocytes, and FPF (Fig. 1Go, A and B). Because POMC is processed by PC1 and PC2 in association with the regulatory protein 7B2, the expression of these enzymes at the message level was also analyzed in cultured HFM, HFK, and FPF and produced the expected 674-bp (PC1), 299-bp (PC2), and 454-bp (7B2) products (Fig. 1Go, C–E). The bands for MC-1R, POMC, and the processing enzymes were discrete and reproducible, and used the precise primer pairs confirmed in previously published protocols (52, 53, 54) for MC-1R, POMC, and the processing enzymes, respectively. Amplification of RNA samples that were not reverse transcribed and samples from which cDNA was omitted from the reaction mixture were also subjected to PCR. These negative controls did not reveal a specific product (Fig. 1Go). Thus, cultured follicular melanocytes express mRNA for MC-1R, POMC, and the PCs.



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  		FIG. 1. Detection of POMC-, MC-1R-, PC1-, PC2-, and 7B2-specific transcripts by RT-PCR in cultured follicular melanocytes, keratinocytes, and follicular papilla fibroblasts. Shown are detection of a 260-bp product specific for POMC (A), detection of a 416-bp product specific for MC-1R (B), detection of 674-bp product specific for PC1 (C), detection of a 299-bp product specific for PC2 (D), and detection of a 454-bp product specific for 7B2 (E). A: Lane 1, DNA ladder; lane 2, HFM; lane 3, HFK; lane 4, FPF; lane 5, plasmid containing the POMC gene; lane 6, negative control 1, omission of cDNA; lane 7, negative control 2, substitution of cDNA with total RNA. B, As above, but in lane 5, plasmid contained MC-1R gene. C: Lane 1, DNA ladder; lane 2, HFM; lane 3, HFK; lane 4, FPF; lane 5, EM (positive control); lane 6, negative control 1, omission of cDNA; lane 7, negative control 2, substitution of cDNA with total RNA. D, As above in C. E, As above in C.

 
POMC peptides and POMC processing machinery are expressed in a subpopulation of human follicular melanocytes in situ
The expression of {alpha}-MSH and ACTH peptide was prominent in the hair bulb matrix and outer root sheath (ORS) of anagen VI hair follicles. Marked expression of both of these POMC peptides was also observed in FP. Individual dendritic cells located in the most proximal and peripheral bulb matrix regions showed strong {alpha}-MSH and ACTH immunoreactivity. The melanocyte identity of these {alpha}-MSH- and ACTH-positive cells was confirmed by double immunolabeling with antibodies against the melanocyte lineage-specific markers, gp100, {alpha}-MSH (Fig. 2AGo), and ACTH (Fig. 2BGo). Interestingly, gp100-positive melanocytes located in the melanogenic zone of the hair bulb populated by melanocytes that are actively engaged in pigment production were negative for these POMC peptides. To assess whether POMC processing machinery was present in human HFM in situ, the expression of PC1, PC2, and 7B2, the regulatory protein for PC2, was examined. The expression of PC1 (Fig. 2CGo) and PC2 (Fig. 2DGo) was pronounced in the hair bulb matrix and the ORS of anagen VI hair follicles. Although strong immunoreactivity for PC2 was seen in the FP, much weaker expression was evident with PC1. The expression of 7B2 was comparable to that observed with PC2 (data not shown). Melanocytes located in the most proximal and peripheral bulb matrix and those located in the ORS exhibited strong immunoreactivity for the PCs (Fig. 2Go, C and D) and 7B2 (data not shown). Melanogenic bulbar melanocytes were again essentially negative. However, rare PC2-positive melanogenic bulbar melanocytes were detected (Fig. 2DGo).



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  		FIG. 2. Human HFM express POMC peptides ({alpha}-MSH and ACTH), MC-1R, and POMC processing machinery. {alpha}-MSH and ACTH peptides, MC-1R, and POMC processing machinery were detected in a minor subpopulation of melanocytes located in the proximal/peripheral matrix region and also in the ORS (arrowheads), but were low/absent in melanocytes of the melanogenic zone of anagen VI hair follicles (A–E). Prominent expression for these markers was also detectable in the hair bulb matrix, ORS, and FP, with the exception of PC1 immunoreactivity, which was much weaker in the FP. Red, {alpha}-MSH and ACTH, POMC processing machinery, MC-1R; green, gp100; yellow/orange, colocalization. Scale bar, 50 µm.

 
The expression of MC-1R was prominent in individual cells in the ORS and the proximal hair bulb; high levels of expression were also detected in the FP of anagen VI hair follicles. Similar to {alpha}-MSH, ACTH, and the processing enzymes, rare melanocytes located in the most proximal and peripheral bulb matrix showed strong immunoreactivity, whereas those located in the melanogenic zone were negative (Fig. 2EGo). Omission of the primary antibodies but inclusion of preimmune serum from a secondary antibody host did not produce any specific staining (data not shown).

These results demonstrate that {alpha}-MSH and ACTH in addition to MC-1R and the processing machinery for POMC are expressed only by a subpopulation of human follicular melanocytes located in the proximal hair bulb and mostly outside of the melanogenic zone during anagen VI.

POMC peptides, MC-1R, and POMC processing machinery are differentially expressed in pigmented bulbar and amelanotic HFM
Immunostaining of primary HFM cultures revealed that {alpha}-MSH, ACTH, their receptor (MC-1R), and the processing machinery for POMC (PC1, PC2, and 7B2) were expressed at high levels in amelanotic melanocytes, and their expression was significantly down-regulated in highly pigmented bulbar melanocytes. In highly pigmented melanocytes, the expression of {alpha}-MSH, PC1, PC2, 7B2, and MC-1R was significantly weaker and was confined to the perinuclear region of the cell (Fig. 3Go, A1/A2, B1/B2, C1/C2, D1/D2, E1/E2, and F1/F2), whereas weaker ACTH expression was restricted to the cell membrane (Fig. 3Go, B1/B2). These findings are consistent with their expression pattern detected in situ, where expression was restricted to a subpopulation of less melanogenic bulbar melanocytes and was undetectable in melanocytes located in the melanogenic zone of anagen VI hair follicles.



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  		FIG. 3. Immunocytochemical detection of POMC peptides, MC-1R, and POMC processing machinery in HFM primary cultures. Each frame pair represents the same cell population taken with both fluorescence and brightfield microscopy (e.g. A1 and A2). The expression of {alpha}-MSH (A1 and A2), ACTH (B1 and B2), PC1 (C1 and C2) PC2 (D1 and D2), 7B2 (E1 and E2), and MC-1R (F1 and F2) was variable in primary HFM cultures. The expression of POMC peptides, processing enzymes, and MC-1R was higher in amelanotic melanocytes (arrow) and was significantly down-regulated in terminally differentiated pigmented bulbar melanocytes (arrowhead). The gp100 expression in both amelanotic (arrow) and pigmented (arrowhead) bulbar melanocytes confirmed their melanocyte identity (G1 and G2). The negative control from which the primary antibody was replaced with preimmune serum from secondary antibody host (H1 and H2). Scale bar, 17 µm.

 
In amelanotic melanocytes, the expression of {alpha}-MSH, the processing enzymes, and 7B2 was present throughout the cell body and showed a granular distribution pattern with higher levels of immunoreactivity detectable in the perinuclear region and notably also in nuclear structures of the cell. However, weaker and more diffuse immunoreactivity was evident along the melanocyte dendrites (Fig. 3Go. A1 and C1–E1). By contrast, ACTH immunoreactivity was marked on the cell membrane, and more diffuse staining was detected throughout the melanocyte cell body (Fig. 3BGo1). Less defined granular MC-1R immunoreactivity was also located predominately around the perinuclear region (Fig. 3FGo1). This could be due to low receptor numbers in follicular melanocytes or to the detection system employed. Therefore, the expression of the receptor was also examined using a modified labeled avidin-biotin technique. The use of an amplification step makes this more sensitive than the indirect immunofluorescence method. Distinctive surface granular expression was detected, especially in the perinuclear region of the cell and less intensely along the melanocyte dendrites (Fig. 4AGo).



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  		FIG. 4. Detection of MC-1R expression in established follicular melanocyte cultures. MC-1R immunoreactivity was detection of by a modified labeled avidin-biotin technique and showed that receptor expression was prominent around the perinuclear region of the cell, exhibiting a granular distribution pattern. A, Weaker expression was detected along the melanocyte dendrites. B, The gp100 expression in HFM (passage 4) confirmed their melanocyte identity. C, Negative control in which the primary antibody was replaced by preimmune serum from secondary antibody host. Scale bar, 13 µm (A), 17 µm (B and C).

 
The positive gp100 staining in both amelanotic and melanotic melanocytes (Fig. 3Go, G1–G2, and Fig. 4BGo) confirmed the melanocyte identity of the cells and also verified that the down-regulation of expression of the POMC peptides and POMC processing machinery observed in the pigmented bulbar melanocytes was not due to the quenching of fluorescence by the high levels of melanin in these cells. No specific staining was observed in the negative controls in which the primary antibody had been omitted and replaced by preimmune serum from a secondary antibody host (Fig. 3Go, H1–H2, and Fig. 4CGo).

These results demonstrate that human HFM have the capacity to synthesize and process POMC peptides in vitro. Furthermore, the expression of {alpha}-MSH, ACTH, MC-1R, PC1, PC2, and 7B2 is inversely correlated with pigmentation level in primary HFM cultures.

Detection of POMC peptides and POMC processing machinery in HFK cultures
Immunostaining of cultured HFK revealed that {alpha}-MSH, ACTH, and POMC processing machinery (PC1, PC2, and 7B2) were expressed in cultured follicular keratinocytes. Expression of the above markers was variable between cells and appeared to be higher in keratinocytes with a basal cell phenotype compared with more differentiated keratinocytes (Fig. 5Go, A–E). The greatest immunoreactivity for {alpha}-MSH, ACTH, PC1, PC2, and 7B2 was restricted to the perinuclear region of the cell, where it also exhibited a granular distribution pattern. The expression of MC-1R was particularly granular in distribution and was detected predominately on the cell surface (Fig. 5FGo). Cell identity was confirmed by positive staining with the anti-HFK-specific antibody, AE13 (Fig. 5GGo). No staining was seen in the negative control where the primary antibody was omitted and replaced by preimmune serum from the secondary antibody host (Fig. 5HGo). These results demonstrate that human HFK have the capacity to synthesize and process POMC peptides in vitro.



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  		FIG. 5. Cultured follicular keratinocytes express POMC peptides and POMC processing machinery. The expression of {alpha}-MSH, ACTH, PC1, PC2, 7B2, and MC-1R was variable in cultured HFK (passage 2). Expression was higher in keratinocytes with a basal cell phenotype (arrowhead) compared with more differentiated keratinocytes (arrow). A–F, Strong expression was confined to the perinuclear region, where it showed a granular distribution pattern. G, Positive staining was observed with the anti-HFK-specific antibody, AE13. H, Negative control in which the primary antibody was replaced with preimmune serum from secondary antibody host. Scale bar, 20 µm (A–E, G, and H), 10 µm (F).

 
Human FPF have the capacity to synthesize and process POMC
The expression of {alpha}-MSH, ACTH, POMC processing machinery (PC1, PC2, and 7B2), and MC-1R was detected in cultured FPF. However, the highest levels of expression were confined to a subpopulation of cells. In this highly positive subpopulation of cells, the expression of the above markers was observed throughout the cell body, but was stronger in the perinuclear region of these cells. The expression pattern of these markers was granular (Fig. 6Go, A–F). Cell identity was observed with the positive control antibody against vimentin, an intermediate filament protein found in mesenchymal cells (Fig. 6GGo). No specific staining was seen in the negative control, where the primary antibody had been omitted and replaced by preimmune serum from the secondary antibody host (Fig. 6HGo). These results demonstrate that cultured FPF express {alpha}-MSH, ACTH, MC-1R, and POMC processing machinery in vitro.



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  		FIG. 6. POMC peptides and POMC processing machinery are expressed in follicular papilla fibroblasts. The expression of {alpha}-MSH, ACTH, PC1, PC2, and 7B2 was variable in cultured FPF, with the highest levels of expression confined to a subpopulation of cells. A–E, Expression of the above markers was strongest in the perinuclear region of the cells, where it was distributed in a granular pattern (arrowheads). F, Likewise, the expression of MC-1R exhibited a distinctive granular distribution pattern that was marked in a subpopulation of cells. G, Positive staining was observed with the positive control antibody against vimentin, an intermediate filament protein found in mesenchymal cells. H, Negative control in which the primary antibody was replaced with preimmune serum from secondary antibody host. Scale bar, 30 µm (A–D, G, and H), 10 µm (F).

 
{alpha}-MSH and ACTH peptides modulate HFM dendricity in vitro
The functional significance of the in situ and in vitro detection of {alpha}-MSH and ACTH peptides and their receptor in HFM was assessed by determining the effects of Ac-{alpha}-MSH and ACTH1–17 peptide stimulation on follicular melanocyte phenotype. Before {alpha}-MSH and ACTH stimulation, HFM maintained in basal medium exhibited a predominately bipolar morphology with occasional tripolar cells (Fig. 7Go, A and B). Stimulation with Ac-{alpha}-MSH (10–8 M) and ACTH1–17 (10–8 M) variably increased cell dendricity in all cell lines examined (Fig. 7Go, A and B). Ac-{alpha}-MSH appeared to be more potent at stimulating follicular melanocyte dendricity (Fig. 7Go, A1 and A2) compared with ACTH1–17 (Fig. 7Go, B1 and B2). Thus, exogenous {alpha}-MSH and ACTH can significantly increase dendricity in some HFM cultures.



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  		FIG. 7. HFM dendricity is stimulated by Ac-{alpha}-MSH and ACTH1–17. HFM cultures (passage 2–5) were established in basic fibroblast growth factor/endothelin-1 (n = 8)-supplemented medium. FCS and BPE were omitted from the culture medium 48 h before stimulation. A marked increase in cell dendricity was seen 72 h after Ac-{alpha}-MSH and ACTH1–17 stimulation in melanocytes derived from brown hair (A1 and A2) and black hair (B1 and B2). Ac-{alpha}-MSH (A1 and B1) produced a greater increase in melanocyte dendricity compared with ACTH1–17 (A2 and B2). Scale bar, 50 µm.

 
{alpha}-MSH and ACTH stimulate melanogenesis and proliferation in follicular melanocytes in vitro
Studies of the melanogenic and proliferative effects of Ac-{alpha}-MSH and ACTH1–17 stimulation on HFM cultures derived from individuals with hair colors ranging from light brown to black, showed that Ac-{alpha}-MSH significantly increased melanogenesis in all cell lines tested (n = 7; P < 0.001; Fig. 8AGo and Table 1Go). The effects were variable and ranged from 10.5–37.4%, with an average increase of 25.1% (SEM, 3.40%) above control unstimulated levels for Ac-{alpha}-MSH (Table 1Go). ACTH1–17 stimulation appeared to be more effective at stimulating melanogenesis compared with Ac-{alpha}-MSH, producing increases that ranged from 7.5–46.5%, with an average increase of 31.2% (SEM, 4.98%) above control unstimulated levels (Table 1Go). Visible increases in melanogenesis were evident in stimulated cell pellets of cell lines that had low basal melanin levels (Fig. 8AGo1). This was less apparent in HFM lines with high basal levels of melanin (Fig. 8AGo2). The melanogenic effects of Ac-{alpha}-MSH and ACTH1–17 appeared to be marked in HFM derived from darker haired donors (dark brown/black) compared with lighter haired donors (light/mid-brown; Table 1Go).



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  		FIG. 8. Melanogenesis and proliferation in follicular melanocytes is stimulated by Ac-{alpha}-MSH and ACTH1–17. Melanin content was determined spectrophotometrically (475 nm) after sodium hydroxide solublization. Cells with low basal melanin levels showed visible increases in melanogenesis after Ac-{alpha}-MSH and ACTH1–17 stimulation (A2). This visible change was not evident in cells with high basal melanin levels (A2). Cell proliferation was assessed by determining cell counts before and after Ac-{alpha}-MSH and ACTH1–17 stimulation. Results are expressed as the percent increase in melanin content (A) and cell number (B) over control unstimulated levels and are the mean ± SEM of seven cell lines. Statistical significance was assessed by one-way ANOVA: ***, P < 0.001.

 

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  		TABLE 1. Effect of acetylated {alpha}-MSH and ACTH1–17 on melanin content in cultured hair follicle melanocytes

 
Assessment of cell proliferation showed a significant increase in HFM number with Ac-{alpha}-MSH (10–8 M) and ACTH1–17 (10–8 M) stimulation in all cell lines examined (n = 7; P < 0.001; Fig. 8BGo). Similar to melanogenesis, the effects of Ac-{alpha}-MSH and ACTH1–17 stimulation on HFM proliferation were also variable. The Ac-{alpha}-MSH-induced increase in cell number ranged from 8.8–39.8%, with an average increase of 18.6% (SEM, 4.39%) above control unstimulated levels (Table 2Go). The ACTH1–17-stimulated increase in cell number ranged from 10.8–31.4%, with an average increase of 17.7% (SEM, 3.54%) above control unstimulated levels (Table 2Go). Based on the above in vitro results, both Ac-{alpha}-MSH and ACTH1–17 have significant melanogenic and mitogenic effects on HFM in vitro.


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  		TABLE 2. Effect of acetylated {alpha}-MSH and ACTH1–17 on cell number in cultured hair follicle melanocytes

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that human scalp HFM have the capacity to synthesize and process POMC. The machinery for POMC processing (PC1, PC2, and 7B2) together with the POMC peptides ({alpha}-MSH and ACTH) and MC-1R are expressed in human HFM at the message in vitro and at the protein level both in situ and in vitro. Moreover, {alpha}-MSH and ACTH peptides are modifiers of human follicular melanocyte phenotype and can stimulate melanogenesis, dendricity, and proliferation in these cells.

The expression of {alpha}-MSH and ACTH peptides, POMC processing machinery, and MC-1R in normal human epidermis is well documented (8, 18, 57, 58, 59). {alpha}-MSH and ACTH peptides together with POMC processing machinery have also been identified in EM, both in situ and in vitro (59). We have recently shown that ß-END and its high affinity µ-opiate R are expressed in human HFM and that this system is expressed as a function of their anatomical location within the hair follicle and of their differentiation status during the hair growth cycle in situ (46). In human hair follicles, ACTH immunoreactivity has been reported in keratinocytes of the hair bulb matrix and the ORS in human scalp skin (8), and prominent MC-1R expression has been demonstrated in the ORS of human hair follicles (58, 60). However, expression of the {alpha}-MSH-ACTH/MC-1R and POMC processing system (PC1, PC2, and 7B2) in human HFM is poorly understood.

In the current study we demonstrate that POMC in association with the machinery for POMC processing and MC-1R are expressed at the mRNA level in cultured human follicular melanocytes, keratinocytes, and FPF. These findings are in agreement with our previous detection of POMC mRNA in these hair follicle cell subpopulations (46) and with the detection of the POMC-processing enzymes and MC-1R in EM and dermal fibroblasts (54, 61, 62).

Here we present evidence that {alpha}-MSH, ACTH, POMC processing machinery, and MC-1R are differentially expressed as a function of melanocyte location and differentiation status within the full anagen (VI) hair follicle. Immunoreactivity for the above peptides was detected in a subpopulation of melanocytes located in the ORS and in melanocytes located in the most proximal and peripheral matrix region of the hair bulb. Interestingly, gp100-positive melanocytes located in the melanogenic zone (located above and around the upper pole of the FP), the site of active melanin synthesis, were negative for {alpha}-MSH and ACTH, POMC processing machinery, and MC-1R. This is in marked contrast to the expression of ß-END; here melanogenically active melanocytes showed strong ß-END immunoreactivity (46). Interestingly, both the ß-END/µ-opiate R and the POMC/MC-1R system are expressed in the less differentiated melanocyte population located in the proximal bulb matrix of anagen VI hair follicles. This coexpression may suggest some degree of cross-talk between these two systems in this very specific melanocyte subpopulation. The pattern of {alpha}-MSH and ACTH expression observed in both bulbar melanocyte subtypes in the present study is similar to that reported for CRH and CRH receptor-1 (63), the chief regulator of pituitary POMC gene expression and the production and secretion of POMC peptides (5, 64).

Our results indicate that the synthesis and processing of POMC to {alpha}-MSH and ACTH is confined to a minor subpopulation of follicular melanocytes located in the ORS and proximal hair matrix. The role of these melanocytes is unclear, but they may reflect a less differentiated pool of melanocytes involved in reconstruction of the hair follicle pigmentary unit (65). The absence of the {alpha}-MSH-ACTH/MC-1R system in the melanogenic zone suggests that this system is not directly involved in the maintenance of melanogenesis during anagen. The situation in humans therefore may differ from that in mice, where the {alpha}-MSH-ACTH/MC-1R system is considered to be the main regulator of follicular pigmentation. In this species the expressions of {alpha}-MSH (37), ACTH (66), MC-1R (37), and POMC processing machinery (38) show significant hair cycle-dependent fluctuations, being highest in anagen and coinciding with the onset of follicular melanogenesis. In addition, higher levels of PC1 immunoreactivity were reported in murine anagen VI hair follicles compared with PC2 expression (38). The lowest levels of the POMC/MC-1R system and POMC processing machinery were detected during the catagen and telogen phases of the murine hair growth cycle. By contrast, the expression of {alpha}-MSH, ACTH, and POMC processing machinery in the present study did not appear to decline during the human hair growth cycle (data not shown), although stronger PC2 expression was observed in anagen VI FP compared with PC1 expression. Rare {alpha}-MSH- and ACTH-positive melanocytes were detectable in the regressing epithelial strand of catagen follicles and in the epithelial sac of telogen follicles (data not shown). These are likely to reflect apoptosis-resistant melanocytes of the previous proximal anagen bulb (65, 67, 68, 69). Therefore, on the basis of our current findings, the expression of {alpha}-MSH and ACTH may be associated with the ability of some hair bulb melanocytes to survive the apoptosis-driven catagen process.

The major focus of the present study was to examine the involvement of the {alpha}-MSH-ACTH/MC-1R system in the regulation of human HFM biology using an in vitro model. This ligand(s)/receptor system is considered to be an important regulator of human skin pigmentation (14, 15, 16, 17, 18, 19, 20) and of follicular pigmentation/hair cycling in mice (32, 33, 37, 66). Immunocytochemical detection of {alpha}-MSH and ACTH, MC-1R, and POMC processing machinery in HFM primary cultures (which consist of both melanogenic bulbar melanocytes and amelanotic melanocytes) revealed that expression levels were inversely correlated with the degree of melanization. Strongest expression was evident in proliferating/differentiating melanocytes containing little melanin. These findings are consistent with the pattern of expression observed in situ, where the expression of the above markers was restricted to a subpopulation of less melanogenic melanocytes located in the most proximal and peripheral matrix of the anagen VI bulb. Melanogenesis in EM is considered to be principally regulated via an MC-1R-dependent mechanism; our current observations suggest that melanogenesis in the human hair follicle is regulated via an MC-1R-independent mechanism. {alpha}-MSH/ACTH and MC-1R are only expressed in relatively undifferentiated melanocytes of the ORS and the proximal/peripheral matrix; {alpha}-MSH-ACTH/MC-1R signaling may be involved the biology of these undifferentiated melanocytes. The current results suggest that POMC peptides such as {alpha}-MSH and ACTH may have greater relevance in the biology of ORS/proximal hair bulb matrix melanocytes than those in the melanogenic zone. For example, this signaling system may participate in migration of the melanocytes into the hair matrix and then with their subsequent differentiation into pigment-producing cells. In humans, the evidence indicates that ACTH may stimulate and/or prolong anagen, because overproduction of ACTH or therapeutic administration of ACTH causes acquired hypertrichosis associated with increased pigmentation (70), suggesting the involvement of ACTH in stimulating melanocyte differentiation and, hence, melanogenic activity.

In addition to describing the full expression of the {alpha}-MSH-ACTH/MC-1R system, we also provide direct evidence for the existence of a functionally active {alpha}-MSH-ACTH/MC-1R system in human HFM. Stimulation of follicular melanocyte cultures with Ac-{alpha}-MSH and ACTH1–17 increased melanogenesis, dendricity, and proliferation, and these effects were comparable to those observed with ß-END stimulation of EM and HFM (46, 55) and {alpha}-MSH and ACTH stimulation of EM (16). Parameters associated with melanocyte differentiation include increased pigmentation and dendricity; both of these were up-regulated after Ac-{alpha}-MSH and ACTH1–17 stimulation in a relatively undifferentiated hair follicle-derived melanocyte population in vitro (i.e. ORS/hair bulb matrix sources). This suggests an important role for these POMC peptides in the differentiation of HFM subpopulations.

This study provides evidence that Ac-{alpha}-MSH and ACTH1–17 are also mitogenic in cultured follicular melanocytes, an effect not normally associated with differentiated cells. This stimulation of both melanogenesis and proliferation in EM by {alpha}-MSH and ACTH has been previously reported (17, 71). Our in vitro studies were conducted using a somewhat heterogeneous HFM cell population. Thus, it may not be immediately clear whether the proliferating cells are also partially or more fully differentiated. The current results also show that ACTH1–17 is more effective at inducing melanogenesis in follicular melanocytes compared with Ac-{alpha}-MSH, whereas Ac-{alpha}-MSH was the most potent at inducing melanocyte dendricity, supporting recent observations that ACTH1–17 is more potent than Ac-{alpha}-MSH at activating MC-1R and stimulating epidermal melanogenesis (18, 72).

The melanogenic and dendritogenic effects of Ac-{alpha}-MSH and ACTH1–17 stimulation in follicular melanocytes appeared to correlate with hair color; a similar association was seen with ß-END stimulation of follicular melanocytes (46). HFM derived from dark-haired individuals (dark brown and black) were the most responsive to Ac-{alpha}-MSH and ACTH1–17 stimulation compared with those from hair follicles of a lighter color. In support of this, it has been shown that {alpha}-MSH-binding sites may be linked to hair color (73); high numbers of {alpha}-MSH-binding sites were detected on human scalp hair bulbs derived from pigmented hair, and fewer binding sites were demonstrated on hair bulbs derived from blond hair and were absent from bulbs derived from senile white hair.

Prominent expression of POMC peptides, its processing machinery (PC1, PC2, and 7B2), and MC-1R in hair follicle epithelial compartments and in the FP indicates that these hair follicle components may be local sources of POMC peptides that could regulate melanocyte behavior via paracrine mechanisms, especially those melanocytes located in the most proximal and peripheral hair bulb matrix. Furthermore, HFK and FPF cell subpopulations can synthesize and process POMC in vitro and also express MC-1R. These findings are in agreement with the detection of {alpha}-MSH, ACTH, and POMC processing machinery in the epidermis (18) and dermis (54). A role for {alpha}-MSH and ACTH peptides in cell differentiation in this study is indicated by the correlation of POMC peptide expression and its processing machinery with follicular keratinocyte differentiation status.

The responsiveness of EM to {alpha}-MSH and ACTH stimulation is thought to reflect differences in the extent of peptide binding to MC-1R and its subsequent signaling activity. Unresponsiveness to these peptides is reported in EM derived from individuals with red hair and fair skin (16, 74). Such control points that modulate ORS/hair bulb matrix melanocyte differentiation into pigment-producing cells may operate in the human hair follicle. Furthermore, MC-1R polymorphisms associated with red hair and fair skin in northern Europeans have also been reported in red-haired individuals with African ancestry (75). However, a pale complexion is not observed in these individuals, suggesting that MC-1R polymorphisms may have greater effects on hair color than on skin color.

In humans, distinct body site variation in hair color is observed; for example, some males exhibit eumelanogenic scalp hair and have pheomelanogenic beards. By comparison, little difference is seen in the epidermis. In mice, coat color is also regulated by ASP, which is produced locally in the FP and acts in a paracrine manner to antagonize the effects of {alpha}-MSH (76, 77, 78, 79). A similar role may exist for ASP in the human hair follicle, which may be responsible for the site-specific variation in hair color. Furthermore, attractin has been proposed to function as an additional receptor for ASP signaling in mice (80, 81, 82, 83). Thus, our finding that {alpha}-MSH, ACTH, and MC-1R expression was undetectable in melanogenically active hair bulb melanocytes and the recent detection of ASP gene polymorphism strongly associated with dark hair and brown eyes (84) suggest that the regulation of human pigmentation does not reside exclusively at MC-1R. Human hair pigmentation may be additionally regulated by the combined effects of other systems such as ß-END/µ-opiate R and ASP/attractin signaling.

In conclusion, we have shown that the human scalp hair follicle contains several melanocyte subpopulations that can be additionally discriminated on the basis of their POMC peptide and MC-1R expression profiles and, importantly, their differential responsiveness to these peptides in vitro. The POMC/MC-1R system appears to be expressed most markedly only during early stages of melanocyte differentiation and becomes down-regulated in mature and fully melanogenic melanocytes. These findings suggest a role for {alpha}-MSH and ACTH peptides in regulating human HFM differentiation. Thus, the maintenance of actual melanogenesis and dendritic phenotypes in hair bulb melanocytes may be regulated by other POMC-associated/nonassociated systems. It is likely that pigmentation is not the only role of follicular melanocytes; thus, POMC peptides, such as {alpha}-MSH and ACTH, may also be important in other aspects of HFM biology.


   Footnotes
 
This work was supported by funding from Procter and Gamble Ltd. (United Kingdom).

First Published Online October 21, 2004

Abbreviations: Ac-{alpha}-MSH, Acetylated {alpha}-MSH; ASP, agouti signaling protein; BPE, bovine pituitary extract; ß-END, ß-endorphin; EM, epidermal melanocyte; EMEM, Eagle’s MEM; FCS, fetal calf serum; FP, follicular papilla; FPF, follicular papilla fibroblast; gp100, glycoprotein 100; HFK, hair follicle keratinocyte; HFM, hair follicle melanocyte; MC-1R, melanocortin-1 receptor; µ-opiate R, µ-opiate receptor; ORS, outer root sheath; PC, prohormone convertase; POMC, proopiomelanocortin.

Received August 27, 2004.

Accepted for publication October 15, 2004.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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